1. Field of the Invention
This invention relates generally to immunoassays (particularly solid-phase fluorescent immunoassaysxe2x80x94SPFIAs), to devices for detecting analytes by immunoassay using capillary tubes, to an apparatus for use with such devices, and to manual, semi-automated, and automated, methods for such testing.
2. Background of the Invention
Many situations exist where qualitative, semi-quantitative, or quantitative detection of the presence of an analyte in a sample is desired. Situations where analyte detection is desirable arise in diverse industries, including: 1) the health care industry, e.g., in clinical and diagnostic medicine (e.g., in vitro analysis); 2) the food processing and chemical industries, e.g. in quality control for food production; and 3) the environmental control industry, e.g. monitoring for the presence of various pollutants in air, ground water, or soil.
Many assays using unique devices and protocols to detect the presence of analytes through chemical and physical means have been developed. Immunoassays make up one broad field of assays which find use in the detection of analytes. In immunoassays, the occurrence of binding events between specific binding pair members is used as an indication of the presence of analyte in the sample. Benefits of using immunoassays, as compared to non-immunoassays, in analyte detection include high sensitivity, high specificity, reliability, and relatively short assay times.
The binding events that are utilized in immunoassays often occur at the surface of a solid support with one binding member held at the surface of the solid support and the other binding member in the sample. The time required for a particular immunoassay to be completed will depend on the ability of the binding member in the sample to reach and bind to the member on the support surface. The ability of the binding member in the sample to bind with its pair on the support surface is dependent on many factors; such factors include the concentration of the binding member on the support surface, and the surface to volume ratio of the sample/support combination. One method to decrease the time required for an immunoassay is to increase the concentration of a binding member on a support surface. Another approach is to increase the ratio of the surface area of the support relative to the volume of the sample to be assayed.
Common immunoassays include radio immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), and membrane based assays, such as common home pregnancy tests. These immunoassays have several disadvantages. A significant disadvantage of RIA is the requirement for the use of hazardous radioactive isotopes. A disadvantage of ELISA is the numerous steps of sample addition, incubation, washing, addition of color reagent, addition of stop reagent, and reading required to perform the assay; such manipulations can be especially troublesome and a source of significant error in the field. Also, enzyme reactions tend to be temperature sensitive, which require temperature control. Unlike RIA, in an which the label is directly detected, in an ELISA the enzyme label is not directly detected. Instead, one must allow for a detectable product to be produced. Further, ELISA protocols may not be suited for all assays on all types of liquids, such as where the liquid comprising the analyte of interest contains contaminants which interfere with one or more individual steps in the assay, e.g. enzyme activity, detectability of enzyme product, and the like. Additionally, the safety concerns with RIA and the complexity of ELISA typically require that they be performed by relatively highly trained personnel and further require constant monitoring by or interaction with the trained operator. A disadvantage with membrane based assays is that they often provide poor quantitation and sensitivity. Many of these assays also require long incubation times, typically in the tens of minutes to hours, making analysis of multiple-samples time consuming and expensive.
Nevertheless, ELISA is a commonly used format. In ELISA, binding events of interest are detected through the appearance of detectable product produced by an enzyme acting on a substrate. The formation of the detectable product can be amplified to the extent required by increasing the concentration of the substrate and/or increasing the reaction time. On this basis, there is an opportunity to greatly increase the signal when only a few enzymes becoming bound.
Conventionally, ELISA has been conducted in microtiter plates consisting of wells. In an effort to improve performance, ELISA has been demonstrated in capillary tubes. With ELISA immunoassays conducted in capillary tubes, rapid quantitative results are reported. At least one reported ELISA is described as sensitive and able to detect small amounts of analyte. See e.g. Chandler et. al., xe2x80x9cA new enzyme immunoassay system suitable for field use and its application in a snake venom detection kitxe2x80x9d Clinica Chimica Acta, 121:225-230 (1982). However, the aforementioned disadvantages inherent in ELISA still exist.
Additionally, while ELISA assays have been performed in capillary tubes in the laboratory, no successful products have been developed. A primary reason for this is the difficulty of bringing several solutions into and out of the capillary tubes and the ability to effectively read the result.
Therefore, there is a significant need for a fast, reliable, accurate immunoassay that requires minimal interaction with the operator. There is also a need for an immunoassay that can screen several similar or different samples sequentially or simultaneously for the same analyte or which can screen for different analytes in the same sample or in a plurality of aliquots of the same sample.
An object of this invention is to provide a simple, semi-automated method for detecting and quantifying an analyte in a sample.
It is a further object of this invention to provide a solid-phase fluorescence immunoassay (SPFIA) that has fewer manipulations than a comparable enzyme-linked immunosorbent assay (ELISA) for detecting an analyte in a sample.
A further object of this invention is to provide a rapid SPFIA that independently and semi-quantitatively or quantitatively assays for a plurality of analytes from a plurality of samples or aliquots from the same sample.
A further object of this invention is to provide a reliable SPFIA that requires minimal manipulation of equipment by an operator performing the immunoassay.
A still further object of this invention is to provide aSPFIA that independently and semi-quantitatively or quantitatively assays for a plurality of analytes from a plurality of samples in less than about 5 minutes.
A still further object of this invention is to provide a unique capillary tube suitable to be used to achieve the aforementioned objects of this invention.
A still further object of this invention is to provide a method for preparing a capillary tube to be used to achieve the aforementioned objects of this invention.
A still further object of this invention is to provide a uniquely-designed cartridge for carrying at least one capillary tube (and preferably more than one) that can be used to achieve the aforementioned objects of this invention.
A still further object of this invention is to provide a tray having a reservoir and a plurality of wells for holding a plurality of aliquots of a sample, which tray can be used in conjunction with the cartridge-held capillary tubes to assist in achieving the aforementioned objects.
A still further object of this invention is to provide an apparatus to be used in conjunction with the cartridge-held capillary tubes and sample tray to perform the SPFIA of this invention and to further assist in achieving the aforementioned objects of this invention.
Other objects will be apparent to one of ordinary skill in the art upon reading the follow specification and claims.
The present invention provides devices for screening for one or more analytes in a sample comprising capillary tubes, a cartridge, and a sample tray which can combine to form a portable and disposable testing kit, and an apparatus and process for screening for one or more analytes, and a method for preparing capillary tubes for use with the method for screening for one or more analytes, that are directed to the disadvantages of the prior art and address heretofore unmet needs previously discussed.
One aspect of the present invention is a cartridge for securely holding a plurality of spaced-apart capillary tubes. The cartridge comprises a frame for holding the tubes in a spaced-apart manner, wherein the frame has a pathway in which each capillary tube can be aligned; and at least one region in the frame to expose at least a portion of each capillary tube so that an electromagnetic signal can contact a portion of each tube.
Another aspect of the present invention is a tray for holding multiple portions of a sample. The tray comprises a reservoir sufficient to hold a quantity of fluid and a shelf extending substantially perpendicularly outward from a sidewall of the reservoir, the shelf having a plurality of spaced-apart wells therein.
Another aspect of the present invention is a process for screening for an analyte in a sample. The process comprises importing a fluid mixture into a capillary tube coated on at least a portion of its interior surface with a substrate, wherein the fluid mixture comprises a sample suspected of containing the analyte and a reagent comprising a fluorescently-labeled conjugate that is (a) capable of binding to the analyte or to the analyte and the substrate and (b) capable of fluorescing when irradiated with an appropriate electromagnetic signal; maintaining the fluid mixture in the capillary tube for a time sufficient for binding to take place between the substrate and the fluorescently-labeled conjugate; removing excess fluid mixture from the capillary tube; externally irradiating the coated portion of the capillary tube with an electromagnetic signal sufficient to cause fluorescence of bound fluorescently labeled conjugate; and detecting the resulting fluorescence to screen for the analyte.
Still another aspect of the present invention is an apparatus for screening for at least one analyte in a sample. The apparatus comprises a reservoir for a fluid; a conduit to transport the fluid to a port; the port being positioned to draw the sample thereto and to pump fluid therethrough; a means to draw at least a portion of the sample to the port; a means to pump the fluid through the port; a first section having connecting means for a cartridge holding at least one capillary tube so that one end of the capillary tube is in fluid communication with the port; a second section having means to hold a tray having at least one well to communicate with the other end of the capillary tube, the second section also having a means to create a changing magnetic field so that a magnetizable metallic object held within the well of the tray is moved sufficiently to agitate a sample when placed in the well; a means to hold the cartridge and capillary tube to permit the capillary tube to be exposed to a signal generation means; the signal generation means; and a signal detection means positioned to detect a signal emitted from the capillary tube as a result of exposure to the signal from the signal generation means.
Another aspect of the present invention is a combination of a cartridge holding at least one capillary tube. The combination comprises a capillary tube coated on at least a portion of its interior surface with a substrate that is capable of binding to a fluorescently-labeled conjugate and a frame comprising a means for positioning the capillary tube in an exposure region of the frame, wherein the exposure region permits exposure of at least a portion of the coated capillary tube to an external electromagnetic signal that is capable of causing bound fluorescently-labeled conjugate to fluoresce.
Another aspect of the present invention is a capillary tube comprising a substrate on at least a portion of its interior surface, which substrate is capable of being bound to a fluorescently-labeled conjugate.
Still another aspect of the present invention is a process for preparing a glass capillary tube for use in a fluorescent immunoassay. The process comprises coating at least a portion of the internal surface of the capillary tube with a substrate that is capable of binding to a fluorescently-labeled conjugate.